Enhancement of protozoan pathogen Perkinsus marinus

Vol. 5: 205-213, 1988 DISEASES OF AQUATIC ORGANISMS Dis. aquat. Org.

Published December 2

Enhancement of protozoan pathogen Perkinsus marinus infections in American oysters

Crassostrea virginica exposed to the chemical carcinogen n-nitrosodiethylamine (DENA)*

James T. Winstead, John A. Couch

US Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, Florida 32561, USA

ABSTRACT: American oysters Crassostrea virginica exposed to high concentrations (600 mg I-') of n- nitrosodiethylamine (DENA) during winter (February to May) showed significant enhancement of an epizootic apicomplexan parasite, Perkinsus rnarinus. The parasite reproduced and caused atypical lesions in exposed oysters in water temperatures at its lower range (20°C). The reasons for this enhancement are not clear but may reflect damage to the oysters' nonspecific, cellular defense mechanisms by the DENA without concomitant negative effects on the parasite.

INTRODUCTION ginica, with the chemical carcinogen DENA. Histo- pathological consequence of the infections and possi-

Perkinsus marinus, formerly Derrnocystidium man- ble reasons for the enhancement phenomenon are also num, is epizootic in American oysters Crassostrea vir- discussed. ginica and a significant cause of mass mortality in Gulf coast and mid-Atlantic estuaries of the USA (Mackin 1951, Ray 1954a,b, Dunnington 1956, Mackin & Hop- MATERIALS AND METHODS kins 1962, Lauckner 1983, Sparks 1985). The tax- onomy, morphology and pathology of P. marinus have American oysters Crassostrea vjrginica were col- been extensively reviewed (Perkins 1976, Levine 1978, lected from East Bay, Santa Rosa County, Florida, USA, Lauckner 1983, Sparks 1985). in November 1986 and held in flow-through seawater

Previous investigators indicated that Perkinsus tanks until experiments were begun in February 1987. marinus infections in oysters are enhanced by tenl- A baseline sample (50 oysters) was taken at the collec- peratures above 20°C (Maclun 1962, Quick & Mackin tion site and 17 individuals were sampled for histologi- 1971, Andrews 1976), salinities above 15 %O (Mackin cal examination 3 d prior to tests. Exposed and control 1956, Scott et al. 1985), close proximity or crowding oysters were held in static 12 l aquaria during each test. (Mackin 1962, Andrews 1965, 1979), and spawning No attempt was made to feed exposed or control oys- state of the oysters (Ray 1954a, Andrews & Hewatt ters during tests. Temperature and salinity were niain- 1957, Mackin 1962, Sinderman 1970). Scavengers tained at 20°C and 20%0, respectively. Expenmental (Hoese 1964) and ectoparasitic snails (White et al. 1987) oysters were exposed to 600 mgl-l of n-nitrosodiethy- which feed on oysters infected with the parasite may lamine (Sigma N0258) for up to 28d. act as vectors in transmitting the disease to other oys- Four separate tests were conducted over a 14wk ters. period (Tables 1 and 2). Test 1 was designed to deter-

This paper reports the first experimentally induced mine acute effects of high concentrations of the car- enhancement of Perkinsus mannus in Crassostrea vir- cinogen on exposed oysters. Samples of 3 exposed and

2 control oysters were taken for histological examina-

Contribution No. 645 of the Environmental Research La- tion at 2 j 4 , l1 and l4 . Additionally1 4 boratory exposed and 3 controls were taken when tests were

O Inter-Research/Printed in F. R. Germany

206 Dis. aquat. Org. 5: 205-213, 1988

Table 1. Crassostrea virginica. Results from tests exposing oysters to DENA (600 mg I - ' ) . In Test 1 , 22 oysters were taken for histological examination during test (+); 23 survivors were transferred to clean seawater and held for 3 wk after test termination

before histological examination ( ' )

Test Date of test No exposed Days exposed No. left at test end Percent sampled at test end with

Perk~nsus marin us enhancement

1 Feb 87 45+ 27 23' 100 2 Mar 87 20 I7 5 100 3 Apr 87 20 28 6 67 4 May 87 20 26 7 86

Table 2. Crassostrea virginica. Control oysters from DENA exposure tests. In Test 1, 15 samples were taken during 27 d (+)

Test Date of test No. of controls Days held No. left at test end Percent sampled at test end with

Perkinsus marinus enhancement

1 Feb 87 25 + 27 10 0 2 Mar 87 20 17 20 0 3 Apr 87 20 28 20 0 4 May 87 20 26 20 0

terminated (27 d). Surviving exposed oysters (23 total) observing 6 microscopic fields on each of 10 slides were transferred to clean seawater and histologically prepared from control oysters and 10 each from examined 3 wk later. exposed and baseline oysters. Values for respective

Tests 2, 3 and 4 were conducted between March and groups of 10 slides were averaged to obtain the infec- May 1987 to verify the Perkinsus marinus enhance- tion values. Light infections averaged 3 observable ment observed in Test l . In each test, 20 oysters were parasites per field, moderate infections averaged 16, exposed to the carcinogen and sampled for histological and heavy infections averaged more than 170. examination after mortalities were greater than 50 %. Test2 was terminated after 17 d with 5 surviving test specimens and Tests3 and 4 were terminated after 28 RESULTS and 26d with 6 and 7 survivors, respectively (see Tables 1 and 2) . Control, oysters were also sampled at Histological examination of baseline oysters sampled the same time exposed oysters were taken. 3 d before tests showed all individuals with healthy

Sampled oysters were grossly examined and l c m digestive gland epithelia and vesicular connecbve thick transverse cuts were taken across the digestive tissue (VCT). Most individuals had light to moderate gland, fixed in Davidsons' fluid (Shaw & Battle 1957) infection5 of Perkinsus marinus in gut epithelia con- and embedded in Paraplast". Tissue was sectioned at comitant with some hemocytic response to the patho- 7,um and stained with Hanis' hematoxylin and eosin gen but no severe histologic damage was observed (Luna 1968). (Fig. 3).

Fig. 1 illustrates the general experimental design Results of the 4 tests are shown in Tables 1 and 2. used to evaluate Perkinsus marinus enhancement in Experimental oysters in Tests 2, 3 and 4 began to have oysters exposed to DENA. Microscopic quantification significant mortalities 17 d postexposure to DENA; only of P. marinus infections in test oysters was done to 18 individuals (30%) survived when the tests were obtain relative prevalencehntensity values. The terminated (Table l ) . In contrast, no control oysters number of parasite stages per field (at 4 0 0 ~ ) i.nfecting died (Table 2). Histological examination of moribund or the outer gut loop epithelia (Fig. 2) was quantified by 'gaper' oysters indicated the probable cause of death to

be heavy Perkmsus marinus infect~ons. Examination of

@ Mention of trade names or commercial products does not exposed and control oysters, after tests were termi- constitute endorsement or recommendation for use nated, showed very heavy infections of P. marinus in

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gut epithelia from 15 of 18 (83 % ) experimental oysters Test 1 had no mortalities in experimental or control (Figs. 4 and 5) and only light infections in gut epithelia oysters. Of 18 exposed and 14 control oysters histologi- of controls (Fig. 6). cally examined 14 d postexposure, 83 '10 of exposed and

Winstead & Couch: Enhancement of infection in oysters exposed to a carcinogen 209

100 % of control oysters showed light infections with the parasite. After 27 d, all exposed oysters sampled (4) showed heavy infections of Perkinsus marinus in gut epithelia identical to Tests2, 3 and 4 (Figs. 4 and 5) while all controls had only light infections of the parasite (Fig. 6).

All exposed oysters with heavy Perkinsus marinus enhancement showed similar histopathological effects. A massive increase of uninucleate meronts and inulti- nucleate schizonts in gut epithelia concomitant with sloughing of epithelia and pathogen into the digestive lumen was observed (Fig. 4). No significant hemocytic infiltration into gut epithelia or foci of these cells beneath the epithelia1 basement membrane was seen (Figs.4 and 5). The lack of a significant hemocytic response to such heavy P. marinus infection in gut epithelia is atypical. Fig.? illustrates a typical hemocytic response in feral oysters with heavy infections of the pathogen in gut epithelia and should be compared with the hemocytic response in DENA-exposed oysters (Figs. 4 and 5). In addition, a massive invasion and replacement of nonciliated digestive tubule epithelia by meronts with no significant hemocytic response to the parasite were observed (Figs. 8 and 9). Control oysters with P, marinus infections in gut epithelia showed typical light to moderate hemocytic responses to the pathogen with localized foci of such cells beneath gut epithelial basement membranes (Fig. 6).

All test oysters showed diminished digestive tubule epithelia, which is normal in bivalves held in static aquaria without food for several weeks. Surviving oysters from Test l , histologically examined 3wk after transfer from DENA exposure aquaria to clean water, showed only light infections of Perkinsus marinus in gut epithelia and healthy nonciliated digestive tubules free of the parasite (Fig. 10).

A summary of data (Tables l and 2) from all tests shows that of 105 exposed oysters, 42 (40 %) died within an average of 24 d after tests began. Of 22 exposed oysters remaining alive and histologically examined at the end of tests, 19 (86 %) showed heavy infections of the pathogen. In contrast, no control oysters died or showed histological evidence of heavy Perkinsus marinus infections during or after tests (Table 2).

DISCUSSION

These tests were designed to determine acute toxici- ty of DENA to oysters for later carcinogen assay tests, and the Perkinsus marinus enhancement discovery was an ancillary observation. High concentrations of the carcinogen (600 mgl-') did not appear to induce the same acute histopathologic effects as reported for other bivalve molluscs exposed to nitrosamines. Examination of DENA-exposed oysters showed no congestion of hemolymph sinuses or branchial blood vessels by blood cells or necrohc lesions in VCT reported in A4ytilus edulis exposed to 100 mgl- ' of dimethylnitrosamine (DMN) for 14 d (Rasmussen 1982). Also, no focal hyper- plasia of gut epithelia accompanied by nodular prolif- erations of basophilic cells described in Unio pictorum exposed to DMN or DENA (400 pgl-l) for 4 wk were observed (Khudoley & Syrenko 1978). The reason oysters exposed to DENA in our tests did not show similar acute effects may reflect differences in species sensltiv- ity or response potential. It may be that nitrosamines are not as toxic to post-spawn oysters in cool water temperatures as to other spawning bivalve species. Oysters have been shown to be more suscephble to xenobiotics during spawning when water temperatures are above 25°C (Scott & Vernberg 1979, Scott e t al. 1985).

Significant mortalities occurred in Tests 2, 3 and 4 but there were none in Test 1. The reasons are not clear and may reflect a variation in tests.

Although some of the lesions of Perkinsus marinus infections or classic 'Dermo disease' described by earlier investigators were observed in exposed oysters, there were significant differences. Classic 'Dermo disease' begins as a chronic disorder and may take months to produce mortality in the host (Menzel & Hopkins 1955). The enhancement phenomenon was acute and caused accelerated mortality in test oysters in less than 3 wk. Histologic examination of oysters with the enhancement phenomenon showed some hemocytic response to P. marinus in gut epithelia and vesicular connective tissues of body and mantle, but the response was atypically light for such heavy infections of this parasite (compare Fig. 7 with Figs.4 and 5). Heavy P. marinus infections in oysters are thought to

Figs. 3 to 6. Crassostrea virginica. Basehne oyster 3 d before tests showing moderate Perlunsus marinus infection in gut epithelia with light hemocytic response beneath gut epithelia basement membrane (arrows). ( ~ 8 6 0 ) . E e Typical heavy infection of P. marinus in gut epithelia of oysters exposed to DENA (600 mgl-l) for 17 to 28d. Note sloughing of parasite and epithelia into gut lumen, moderate hemocytlc response by the oyster and atypical invasion of the parasite into nonciliated digest~ve tubules (arrows). ( ~ 8 6 0 ) . E@. Higher magnification of P. marinus infection in oyster gut epithelia exposed to DENA (600 mgl-l) for l 7 to 28d. Note numerous parasite stages in gut epithelia (arrows) w t h a moderate hemocytic response (H) beneath the gut epithelial basement membrane. ( X 1720). Flg.6 Control oyster from DENA exposure tests with a Light infection of P. marinus in gut epithelia. A moderate hemocytic response to the pathogen concomitant with normal dapedesis by blood cells in gut epithelia is occurring due to starvation stress. Note diminished epithelia of nonciliated digestive tubules with no P. marinus

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Winstead & CIouch: Enhancement of infec :tlon in oysters exposed to a carcinogen 211

cause mortality via systemic invasion and damaging blood sinuses (Mackin 1951, Sparks 1985) but this was not observed in DENA-exposed oysters. A striking difference between classic 'Dermo disease' and pathosis in exposed oysters was a massive invasion and destruction of nonciliated digestive tubule epithelia by the pathogen. This is atypical because nonciliated digestive tubules are not generally invaded and des- troyed by P. rnarinus, like gut epithelia, but become necrotic due to destruction of supporting connective tissue and blood sinuses by the parasite (Mackin 1951, Perkins 1976, Sparks 1985).

All baseline oysters histologically examined before tests had light to moderate infections of the parasite in gut epithelia. This.coincides with reports that the prim- ary portal of entry is gut epithelia (Mackin 1951) and the pathogen can lie dormant in host tissue for months in water temperatures of 20°C or less (Hewatt & An- drews 1954, Ray 1954a, Mackin 1962, Quick & Mackin 1971). Prevalence and intensity of infections increase linearly at temperatures above 18°C (Mackin & Sparks 1962, Quick & Mackin 1971), and the parasite does not readily multiply or cause significant pathosis in oysters below this temperature (Andrews & Hewatt 1957, Andrews 1966). Thus, to date, temperature has been established as the most important environmental factor affecting virulence of Perkinsus rnarinus infections in oysters, with the most intense infections occurring above 25°C (Ray 1954a, h4ackin & Sparks 1962, Ray 1966, Lauckner 1983). However, P. rnarinus did not remain dormant in DENA-exposed oysters but reproduced in large numbers causing pathologic damage and significant mortality (86 % ) in temperatures of 20°C. Conversely, the pathogen remained dormant in control oysters and did not multiply or become pathogenic at the same temperature.

It appears the DENA either directly or indirectly enhanced proliferation and lethality of Perkinsus marinus in exposed oysters since control oysters showed no signs of pathogen intensity enhancement and surviving oysters transferred to clean water showed no mortality or histological evidence of enhancement 3 wk after transfer.

The reasons why Perkinsus marinus was able to multiply in tremendous numbers, infect tissues not generally invaded by the parasite, and become lethal in DENA-exposed oysters at temperatures below its normal pathologic range are not clear. It is possible the DENA 'causes a reduction in the effectiveness of inter- nal defense mechanisms or modifies epithelia1 barriers and biochemical processes' in exposed oysters (Sinder- man 1980). Bivalves do not possess humoral immune factors (antibodies) like vertebrates, but respond to pathogenic agents via nonspecific cellular mechanisms, such as leukocytic infiltration, phagocytosis,

encapsulation and diapedesis (Sparks 1972, Cheng 1973). P. rnarinus does not reproduce readily or become intensely pathogenic in oysters below 20°C in natural environments; however, parasite meronts are capable of enlargement in thioglycollate media at 18'C (Ray 1954a). Also, bivalves (Mytilus edulis) are capable of producing histologically detectable hemocytic responses to chemlcal injury in water temperatures below 15°C (Rasmussen 1982). Because control oysters showed no P. marinus enhancement and the carcinogen did not appear acutely toxic to test oysters, evidence suggests the DENA may have been toxic to the oysters' nonspecific cellular defense mechanisms. Prior studies indicate carcinogenic pesticides [such as organochlonnes (DDT, Dieldrin) and carbamates (Urethan)] reduce phagocytic capacity and viability of macrophages and granular leukocytes in vertebrates (Exon et al. 1987). Also, similar enhancement of a pathogen (Baculovirus) in a crustacean (Penaeus duro- arurn) exposed to a polychlorinated biphenyl (Aroclor 1254) has been reported (Couch & Nimmo 1974, Couch & Courtney 1977). An earlier investigation of Crassostrea virginica exposed to chronic low levels of DDT, Toxaphene and Parathion (Lowe et al. 1971) may be relevant to the present study. The consulting pathologist, Mr Gilbert Pauley, reported heavy P. n7arinus infections in all exposed oysters examined, with atypically light hemocytic response to the pathogen. In contrast, few control oysters examined from the study were infected with the parasite. However, unlike the present study, the exposed oysters were sampled when salinities were very high (29 %o) and water warm (25 "C).

If the resistance-compromising hypothesis is correct it would explain why the pathogen was capable of such tremendous proliferation and lethality in exposed oysters. However, it does not explain why the parasite was able to proliferate in such tremendous numbers at a temperature near its lower range. As already discussed , the most important factor which appears to control Perkinsus marinus infections in oysters is temperature. P. marinus is capable of enlargement in thioglycollate media a t 18 "C but has not been reported to be as active as observed in the DENA-exposed oysters in this study. Another possibility relating to the enhancement phenomenon may be that DENA is capable of stimulating the pathogen's growth directly. Few studies address the direct effects of chemicals on disease agents in aquatic animals (Esch e t al. 1975, Lauckner 1983) and the present study indicates more research is needed. Prior investigators have suggested that certain diseases in economically important estuarine animals may b e caused or enhanced by chemical pollutants in the environment (Sparks 1972, Couch & Nimmo 1974, Fries & Tripp 1976, Overstreet & Howese 1977, Couch

212 Dis. aquat. Org. 5: 205-213, 1988

& Courtney 1977, Sinderman 1980, Couch 1985, Couch & Harshbarger 1985) even though there are few studies which dlrectly link specific pollutants to specific diseases. The DENA enhancement of P. mar lnus infections in Crossostrea virginica appears to substantiate the thesis that chemical enhancement of certain pathogens can occur in some aquatic animals (Couch & Nimmo 1974, Couch & Courtney 1977, Couch & Harsh- barger 1985). This may be relevant in epizootiological areas where P. m a r i n u s causes the greatest mortalities in oysters since some studies indicate prevalence and intensity of some infections to be heavier in estuaries impacted with more chemical pollution (Couch 1985). We do not mean to imply that every host-pathogen/ parasite relationship will be umbalanced in favour of only the pathogen at the expense of the host. In certain cases, it is possible that the parasite may be equally or more vulnerable to chemical influence. Each potential chemical effector must be evaluated on a case-by-case basis (empirically) until more is known about their m.echanisms of action.

The exact mechanisms for the Perk insus mar inus

enhancement phenomenon are not understood, but it is possible that DENA may be toxic to the cells of the oysters nonspecific defense mechanisms, or be capable of stimulating the pathogen's growth directly. Because of the economic importance of Crassostrea virginica in epizootiological areas of P. marinus , further studies should be conducted to determine: (1) What nonspecific cellular defense mechanisms exist in oysters with the capacity to keep P. mar inus pathogenicity suppressed in cold temperatures when this study shows the parasite can be very active in these temperatures? (2) Why is P. m a r i n u s atypically active and pathogenic in oysters in the presence of a stressing agent in water temperatures below its normal pathogenic range? (3) Is DENA affecting the pathogen's growth directly? (4) Are other xenobiotics capable of causing similar P. m a r i n u s

enhancement in oysters in cold or warm water temperatures?

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Responsible Subject Editor: Dr A. K . Sparks; accepted for printing on September7. 1988

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Enhancement of protozoan pathogen Perkinsus marinus